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Debrisoquine 4-monooxygenase and bufuralol 1′-monooxygenase activities in bovine and rabbit tissues
Biochemical Pharmacology, Vol. 43, No. 9, pp. 1911-1919, 1992.
0006-2952/92 $5.00 + 0.00 ~ 1992. Pergamon Press Ltd
Printed in Great Britain.
DEBRISOQUINE 4-MONOOXYGENASE A N D BUFURALOL I'-MONOOXYGENASE ACTIVITIES IN BOVINE A N D RABBIT TISSUES YOSHINORI MATSUO,* KAZUHIRO IWAHASHIt and YOSHIYUKIICHIKAWA Department of Biochemistry, Kagawa Medical School, Miki-cho, Kita-gun, Kagawa 761-07, Japan (Received 3 December 1991; accepted 14 February 1992) tissue distributions of debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities in microsomes from bovine and rabbit tissues were analysed. Debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities were found in liver, and at low levels in cerebral cortex, kidney cortex, lung, small intestine and spleen. Other tissues, such as kidney medulla, adrenocortex, adrenomedulla, blood vessels, thyroid gland, heart, ovary, uterus and testis, showed low levels of bufuralol l'-monooxygenase activity but no detectable debrisoquine 4-monooxygenase activity. The bufuralol/debrisoquine monooxygenase activity ratios were higher in kidney and lung, and lower in cerebral cortex and spleen than in liver. Both monooxygenase activities in several bovine tissues including liver were inhibited strongly by phenylisocyanide (0.1mM) and quinidine (0.5mM), moderately by metyrapone (1 raM), and not at all by KCN (1 mM). NuNs (5 raM) and sodium cholate (0.5% w/v) inhibited debrisoquine 4-monooxygenase activity strongly and moderately, but bufuralol l'-monooxygenase activity moderately and strongly, respectively. No effect of a hydroxyl radical scavenger or of superoxide dismutase on either monooxygenase activity was observed. It was concluded from these results, as well as the NADPH dependency of the reactions, that the two monooxygenase reactions observed in these tissues were catalysed by cytochrome P450s.
Abstract--The
Over 25 drugs and chemicals, including debrisoquine, bufuralol, sparteine and dextromethorphan, are metabolized by cytochrome P450 IID [1-4]. Some members of the cytochrome P450 (P450,) subfamily differing only slightly in amino acid sequence show different substrate specificities [5-8], and are expressed differently in various tissues and at various ages [9, 10]. In rat liver, two members of the P450 IID subfamily, the dbl and db2 proteins, were identified [9]. The former exhibits both debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities (dbl type), and the latter bufuralol l'monooxygenase activity but not debrisoquine 4monooxygenase activity (db2 type) [9]. Similarly, in human liver, the dbl type (P450 buff) and db2 type (P450 bufII) were identified [3, 10]. Therefore, the debrisoquine 4-monooxygenase reaction is catalysed by P450 dbl, while the bufuralol l'-monooxygenase reaction is catalysed by more than two forms of P450. Usually, several P450s are responsible for the metabolism of a certain drug or chemical [11]. However, the substrate specificity of debrisoquine * Corresponding author: Y. Matsuo. Tel. (81) 878-985111; FAX (81) 878-98-7109. t Present address: Department of Neuropsychiatry, Kagawa Medical School, Kagawa, Japan. Abbreviations: P450, cytochrome P450; LKM, liver kidney microsomes; MPTP, 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine; TIQ, tetrahydroisoquinoline; G6P, D-glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase. § Tsuneoka Y, Matsuo Y and Ichikawa Y. Characterization and microheterogeneity of cytochrome P-450 IID subfamily from bovine liver: nucleotide sequence and classification of the members of cytochrome P-450 IID subfamily. Manuscript in preparation.
is marked as it is apparently only metabolized by P450dbl and individuals with defects in the gene for P450db1 were poor metabolizers of debrisoquine [12, 13]. Although bufuralol l'-monooxygenase activity has been measured in several rat tissues, such as liver, kidney, brain, lung and spleen [14,15], the tissue distribution of debrisoquine 4-monooxygenase activity, which is important for the metabolism of Parkinsonism-inducing substances such as 1-methyl4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and tetrahydroisoquinoline (TIQ) [14, 16, 17], has not been reported and could be different from the distribution of bufuralol l'-monooxygenase activity. In this study bovine and rabbit tissues were used because of their size, especially small organs such as adrenal glands and various parts of the brain which renders the preparation of microsomes, the purification and extraction of RNA, and the detection of localized proteins or mRNAs easier. We have cloned and analysed cDNA clones of the P450 IID subfamily from bovine liver, and reported the presence of microheterogeneity§. To study the functions and expressional control of members of the P450 IID subfamily, the tissue distributions of debrisoquine 4-monooxygenase and bufuralol l'monooxygenase activities in bovine and rabbit tissues were examined in this study. MATERIALS AND METHODS
Chemicals. Debrisoquine sulfate, 4-hydroxydebrisoquine sulfate, bufuralol hydrochloride (race- [(tert- butylamino)methyl]- 7-ethyl-2-benzofuranmethanol hydrochloride) and l'-hydroxybufuralol were kindly supplied by Dr H. Fukui (Department
1911
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Y. MATSUO, K. IWAHASHI and Y. ICHIKAWA
of Pharmacology, Medical School, Osaka University). D-Glucose 6-phosphate (G6P) and glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides were purchased from Oriental Yeast Co. Ltd, Japan. N A D P H and phenylmethyl sulfonyl fluoride were purchased from the Sigma Chemical Co. (St Louis, MO, U.S.A.). Superoxide dismutase (SOD) was purchased from Toyobo Co. Ltd, Japan. Materials. The cattle (Holstein-Friesian, 1.5 years old) were obtained from a local slaughterhouse. The rabbits (Japan, white, 6 months old) had been given a normal diet and water ad lib. in the laboratory. Fresh tissues from the animals were kept on ice and transported to the laboratory within 1 hr. They were perfused with ice-cold 0.15 M KCI to remove blood. They were then rapidly and carefully cleared of connective tissue, fat and major blood vessels. Preparation of microsomes. Microsomal fractions from bovine and rabbit tissues were prepared by the method of Mitoma et al. [18]. Care was taken to remove hemoglobin as much as possible, as described previously [19]. Contamination of the microsomes of various tissues by mitochondria was found to be less than 5% on a protein basis, as judged from the succinate dehydrogenase activity [20]. Using bovine serum albumin as a standard, the protein concentrations in microsomal fractions were measured by the biuret reaction method [21] in the presence of 0.25% (w/v) deoxycholic acid, which was added to remove turbidity from the sample. The increased absorption at 540 nm in the biuret reaction due to heine in the test samples was avoided by using a solution containing 3.4% (w/v) NaOH, KI (1 g/500 mL) and potassium sodium tartrate 3 g/ 500 mL). The optical absorption at 25° was measured, using cuvettes of 1 cm optical path, with a Cary recording spectrophotometer, model 17D.
Assaying of debrisoquine 4-monooxygenase and bufuralol l '-monooxygenase activities. The enzymatic reactions and chromatographic conditions reported by Kronbach et al. [10] were slightly modified and used for the activity assays. In the NADPHregenerating system, G6P and G6PDH were used instead of sodium isocitrate and isocitrate dehydrogenase. The reaction mixture, which contained microsomal protein (0.3-2 mg), a N A D P H regenerating system without N A D P H (5 mM G6P, 2.5U G6PDH and 4 m M MgC12) and 0.5mM substrate (debrisoquine or bufuralol concentrations of more than three times the Km values reported for human and rat P450db) in 0.05 M sodium phosphate buffer (pH7.4), was preincubated for 3min at 37°. In the reactions with pancreas microsomes, phenylmethyl sulfonyl fluoride was added to 0.5 mM to inhibit protease activity. The reaction was started by the addition of N A D P H (final concentration, 1.6 mM), followed by aerobic incubation for 60 min at 37°. Under these conditions, the enzymatic reactions were linear with time for at least 80 min and microsomal protein concentrations up to 2 mg/ mL. Only when the presence of a trace of activity was checked, the mixture was incubated for 2 hr at 37° . The final volume of the reaction mixture was 1 mL. The reaction was stopped by the addition of 100/~L of 60% HC104 (w/v). Denatured protein
was removed by centrifugation. The enzymatic production of 4-hydroxydebrisoquine or l'-hydroxybufuralol was measured by HPLC (Waters 510), with fluorescence detection, as described by Kronbach et al. [10]. The column used was of Cosmosil 5C-18 (Nacalai Tesque, 4.6 x 250 mm) for both activity assays. In most cases, the retention times of l'-hydroxybufuralol and 4-hydroxydebrisoquine were 4.2 and 9.5 min, respectively. In some cases, however, overlapping peaks of products had to be separated by adjusting the retention time of 4hydroxydebrisoquine in the range of 9.3 to 18.8 rain. In the inhibition experiment, phenylisocyanide, KCN, NaN3, metyrapone, sodium cholate and quinidine were added to reaction mixtures to final concentrations of 0.1 mM, 1 raM, 5 mM, 1 mM, 0.5% (w/v) and 0.5 raM, respectively. Mannitol, a hydroxyl radical scavenger [22], and SOD were added to a final concentration of 200 mM and 1/~M, respectively. RESULTS
Debrisoquine 4-monooxygenase activity in various tissues Debrisoquine 4-monooxygenase and bufuralol l'monooxygenase activities in the microsomes of various tissues from male and female cattle, and male and female rabbits were analysed. The results are summarized in Table 1. A high level of debrisoquine 4-monooxygenase activity was detected in liver from both cattle and rabbits, and a low level in the cerebral cortex of cattle, lung of cattle and rabbits, small intestine of rabbits, spleen of cattle and kidney cortex of rabbits. No significant sex differences were detected for the debrisoquine 4-monooxygenase activity in the rabbit tissues studied. Debrisoquine 4-monooxygenase activity in male rabbit liver was eight times higher than in male bovine liver (35.5-+ 6.5 vs 4.4 +-0.6nmol/hr/mg protein) The order of activity in rabbit liver was the same as in rat liver [9], and that in bovine liver was the same as in human liver [10, 23]. Debrisoquine 4-monooxygenase activity in bovine cerebral cortex was about 3% of the liver value. Because of the low level, the activity could not be detected when rabbit microsomes from whole cerebrum were analysed as to enzyme activity. None of the other brain parts, i.e. midbrain, cerebellum and medulla oblongata, showed detectable activity. This suggested that debrisoquine 4-monooxygenase activity is higher in the cerebral cortex region of the cerebrum than any other brain parts studied. This is consistent with the regional distribution of spartein monooxygenase activity in the brain [24]. Debrisoquine 4-monooxygenase activity in bovine kidney cortex could not be detected, but low levels of activity, 1.5% and 0.8% of the liver value, were found in male and female rabbit kidney cortex, respectively. In lung, activity levels of 0.7% and 0.5% of the liver value were observed in male cattle and female rabbits, respectively. The activities in small intestine, 0.3% of the liver value, and spleen, 0.6% of the
Tissue distribution of debrisoquine/bufuralol monooxygenase activities
1913
Table 1. Debrisoqnine 4-monooxygenase and bufuralol l'-monooxygenase activities in microsomes of various tissues from bovine and rabbit Debrisoquine 4-monooxygenase (nmol/hr/mg protein) (%)
Bufuralol l'-monooxygenase (nmol/hr/mg protein) (%)
Male Male Female
4.417 -+ 0.551(2) 35.526 - 6.491(2) 23.812
100 100 100
34.397 ± 4.318(2) 66.596 65.729
100 100 100
Bovine Rabbit Rabbit Rabbit Rabbit
Male Male Female Male Female
ND(3) 0.526 0.187 ND(2) ND(2)
1.48 0.79
0.392 -4-0.328(3) 1.653 -*- 1.955(5) 2.933 - 2.007(3) 0.540 - 0.592(2) 0.144 _-+0.172(2)
1.14 2.48 4.46 0.82 0.22
Bovine Bovine
Male Male
ND(3) ND(2)
0.133 ± 0.035(3) 0.075 ± 0.006(2)
0.39 0.22
Bovine Rabbit Rabbit Rabbit Rabbit Rabbit
Male Male Female Male Male Female
0.132 -+ 0.083(3) ND ND ND ND ND
0.085 -+ 0.050(3) 0.018 0.026 0.017 0.010 0.009
0.25 0.03 0.04 0.03 0.02 0.01
Rabbit Bovine Rabbit Bovine Rabbit Bovine Rabbit Rabbit Rabbit Bovine Rabbit Bovine Rabbit Bovine Rabbit Rabbit Rabbit
Male Male Female Male Female Male Male Female Female Male Female Male Female Female Female Female Male
ND 0.032 0.127 ND 0.070 ND ND ND ND ND ND 0.028 ND ND ND ND ND
0.010 0.196 0.843 0.024 0.076 0.005 0.053 0.063 0.038 0.022 0.039 0.067 0.016 0.019 0.017 0.034 0.165
0.02 0.57 1.28 0.07 0.12 0.01 0.08 0.10 0.06 0.06 0.06 0.19 0.02
Tissues
Animals
Liver
Bovine Rabbit Rabbit
Kidney Cortex Medulla Adrenal gland Cortex Medulla Brain Cerebral cortex Cerebrum Midbrain Cerebellum Medulla oblongata Lung Small intestine Blood vessels Thyroid gland Heart Pancreas Spleen Ovary Uterus Testis
Sex
2.99
0.72 0.53 0.29
0.63
0.03 0.05 0.25
Values are means -+ SD. Within parentheses are the number of samples from different animals. ND, not detected; the enzyme activities less than 0.025 and 0.001 nmol/hr/mg protein of microsomes cannot be detected for debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase, respectively.
liver value, were detected in only one of the two species studied, respectively. There was no detectable debrisoquine 4-monooxygenase activity in kidney medulla, adrenocortex, adrenomedulla, blood vessels, thyroid gland, heart, pancreas, ovary, uterus or testis. To check for the presence of a trace of debrisoquine 4-monooxygenase activity in bovine kidney cortex and adrenocortex, the reaction mixtures were incubated longer (2 hr). However, 4-hydroxydebrisoquine, the product of the reaction, could not be detected in these tissues. Low levels of other unknown metabolites were detected in microsomes of the adrenocortex (Fig.
1).
Bufuralol l'.monooxygenase activity in various tissues Bufuralol l'-monooxygenase activities in various tissues are shown in Table 1 and representative
chromatographic patterns in Fig. 2. There was a general tendency that the higher was the debrisoquine 4-monooxygenase activity in certain tissues, the higher was the bufuralol l'-monooxygenase activity in the same tissue. High levels of bufuralol l ' monooxygenase activity were found in liver, and low levels in kidney cortex, adrenocortex, adrenomedulla, cerebral cortex, lung, small intestine and spleen. Traces of activity were found in cerebrum, midbrain, cerebellum and medulla oblongata from rabbit brain, and blood vessels, thyroid gland, heart, pancreas, ovary, uterus and testis. The activity levels in liver from male cattle, and male and female rabbits were 34.4, 66.6 and 65.7 nmol/hr/mg protein, respectively. The bufuralol l'-monooxygenase activity levels in kidney cortex from male cattle, and male and female rabbits were 1.1%, 2.5% and 4.5% of the liver values, respectively.
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Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
4-OH
(A) Bovine Kidney
(C) Bovine Adrenocortex
(a) Rabbit Liver
(D) 4-OH Debrisoquine
F Fig. 1. Chromatograms of debrisoquine metabolites with a longer incubation period (2 hr). (A) Incubation of kidney cortex microsomes from male cattle with debrisoquine. (B) Liver microsomes from male rabbits. 4-OH, 4-hydroxydebrisoquine. (C) Adrenocortex microsomes from male cattle. (D) Standard 10.4 nmol of 4-hydroxydebrisoquine. Six hundred microlitres of the reaction mixture were injected in each case.
Bufuralol l'-monooxygenase activity was found in adrenal cortex and medulla, 0.4% and 0.2% of the liver value, respectively. In adrenal cortex and medulla, no debrisoquine 4-monooxygenase activity was detected, suggesting the presence of db2 type I)450 in adrenal cortex and medulla.
A low level of bufuralol l'-monooxygenase activity was found in cerebral cortex (0.3% of the liver value) and very low levels in other brain parts. Like debrisoquine 4-monooxygenase activity in cerebral cortex, bufuralol l'-monooxygenase activity was also higher in the cerebral cortex region of the brain.
Tissue distribution of debrisoquine/bufuralol monooxygenase activities
1915
1 ' -OH
I'-OH
1 ' -01/
I'-OH
1 '-OH
1 ' -OH
1 ' -OR
(A) I'-OH Bufuralol
(R)
(C)
(D)
Rabbit Liver 1 /zl
Bovine Liver 1 ;tl
Bovine Kidney 100 /.zl
(El Bovine Adrenocortex i00 ~ I
(F)
(G)
Bovlne
Bovine
Adrenoaedulla 200 ~ I
Cerebral 200 /.zl
cortex
Fig. 2. Chromatograms of bufuralol metabolites. (A) Forty picomoles of l'-hydroxybufuralol as a standard. (B) Metabolites formed by liver microsomes from male rabbits. One microlitre of the reaction mixture was injected. 1'-OH, l'-hydroxybufuralol. (C) Liver microsomes from male cattle, 1 #L. (D) Kidney cortex microsomes from male cattle, 100/zL. (E) Adrenocortex microsomes from male cattle, 100 #L. (F) Adrenomedulla microsomes from male cattle, 200/zL. (G) Cerebral cortex microsomes from male cattle, 200 #L.
Other tissues which showed low levels of bufuralol l'-monooxygenase activity were lung, small intestine and spleen. These tissues also showed low levels of debrisoquine 4-monooxygenase activity. No significant sex difference was detected for bufuralol l'-monooxygenase activity in rabbits.
Bufuralol/debrisoquine monooxygenase ratio in various tissues The bufuralol l'-monooxygenase/debrisoquine 4monooxygenase activity ratios are shown in Table 2. There was variation in the ratio in various tissues. -In liver, the ratios were 7.8 in male cattle, 1.9 in male rabbit and 2.8 in female rabbit, however, the ratios in kidney cortex were 3.1 in male rabbit and 15.7 in female rabbit. These values were higher than in liver. In lung the ratio in female rabbit, 6.6, was higher than in liver, but the ratio in male cattle, 6.1, was about the same as in liver. In contrast, bovine cerebral cortex and spleen showed lower ratios, 0.6 and 2.4, respectively, than liver.
Effects of inhibitors on debrisoquine 4-monooxygenase and bufuralol l '-monooxygenase activities To confirm the P450 dependent reaction, the dependence of the reaction on N A D P H as an electron donor, the effects of six chemicals, phenylisocyanide, KCN, NAN3, metyrapone, sodium cholate and quinidine, on the debrisoquine 4monooxygenase and bufuralol l'-monooxygenase activities were investigated with microsomes from several bovine tissues, i.e. liver, kidney cortex, adrenocortex, adrenomedulla and cerebral cortex. Phenylisocyanide is known to be a specific inhibitor of P450 [25] and sodium cholate is a denaturing reagent, converting P450 to P420 [26]. The results are shown in Table 3. Debrisoquine 4-monooxygenase activity in liver was strongly inhibited by 0.1 mM phenylisocyanide, 5 mM NaN 3 and 0.5 mM quinidine to 1.6%, 9.4% and 8.7% of the control value, respectively, and moderately by 1 mM metyrapone and 0.5% (w/v) sodium cholate, to 16.3% and 23.2%, respectively,
Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
1916
Table 2. Ratios of bufuralol l'-monooxygenase (But') to debrisoquine 4-monooxygenase(Deb) activities in various tissues Tissues Liver
Animals
Bovine Rabbit Rabbit Kidney cortex Rabbit Rabbit Brain cerebral cortex Bovine Lung Bovine Rabbit Small intestine Rabbit Spleen Bovine
Sex Male Male Female Male Female Male Male Female Female Male
Ratios (Buf/Deb) 7.79 1.87 2.76 3.14 15.68 0.64 6.13 6.64 1.09 2.39
Effects of a hydroxyl radical scavenger and SOD on the enzyme activities To investigate the effects of hydroxyl radicals and OF, a hydroxyl radical scavenger, mannitol, and SOD were added to final concentrations of 200 mM and 1/~M, respectively, and then the enzyme activities in several bovine tissues were measured (Table 3). Although debrisoquine 4-monooxygenase activity in liver in the presence of mannitol was decreased to 70%, bufuralol l'-monooxygenase activity did not change, 82-107% of the control value. SOD did not affect the enzyme activities, 97114% of the control values, in the tissues studied. These results show that neither hydroxyl radicals nor O~ had significant effects on these activity assays. DISCUSSION
but not inhibited by 1 mM KCN. The reaction without NADPH or a substrate showed no detectable debrisoquine 4-monooxygenase activity. A similar activity inhibition pattern was seen in cerebral cortex. Because of the low activity in cerebral cortex, which was just above background, the activity inhibited by phenylisocyanide, NAN3, metyrapone and sodium cholate could not be detected. The activity was not inhibited by KCN, 90.5%, and not detected without NADPH or a substrate. Bufuralol l'-monooxygenase activities in microsomes from the various tissues were inhibited very similarly by these inhibitors, except for cerebral cortex. The activities of various tissues were inhibited strongly by 0.1 mM phenylisocyanide, 0.5% (w/v) sodium cholate and 0.5 mM quinidine, 4-18% of the control value, and moderately by 1 mM metyrapone and 5 mM NaN3, 20--69%. One millimolar KCN did not inhibit the activity, 93-104%. No detectable activity was found without a substrate. However, traces of activity were detected without NADPH, 0.003-0.055 nmol/hr/mg protein, in which case the reaction might be driven by either endogenous NADPH or an enzyme that is independent of NADPH. The activity inhibition pattern in cerebral cortex seemed to be different. However, the activity was inhibited 49.1%, 32.1% and 39.6% by 0.1 mM phenylisocyanide, 5mM NaN2 and 0.5% sodium cholate, respectively, but not at all by 1 mM KCN or 1 mM metyrapone. These results of inhibition experiments, i.e. that the activities in several tissues were strongly inhibited by phenylisoeyanide, inhibited very similarly by several inhibitors and depended on NADPH, confirmed that the low level of the debrisoquine 4monooxygenase reaction in brain, and the bufuralol l'-monooxygenase reactions detected in kidney cortex, adrenal gland and brain, as well as the high level in liver, were catalysed by P450. P450s have been classified into two groups according to their sensitivity to KCN [27, 28]. From the results of the inhibition experiments, it is clear that P450 db belongs to the group insensitive to KCN.
Bufuralol l'-monooxygenase activity has been reported in several rat tissues, such as liver, kidney, brain, adrenal, lung and spleen [15]. The activities were very close to those reported here for cattle and rabbits. Debrisoquine 4-monooxygenase activity in various tissues was also determined and variations in the bufuralol/debrisoquine monooxygenase activity ratio were found. Moreover, the ratios in a single tissue in two species were also different, for example, the level of debrisoquine 4-monooxygenase activity in rabbit liver was eight times higher than that in bovine liver. However, bufuralol l'-monooxygenase activity was not very different in the livers of the two species. The variation in the activity ratio in tissues or species could be explained by either or both of the following. (1) The efficiency of debrisoquine or bufuralol metabolism by a single P450 molecule. (2) The ratio of the expressed proteins, dbl type and db2 type. To clarify this, enzymatic and kinetic parameters, such as Km and Vmax, and the turnover numbers for each type of db protein and the degree of expression of each member of the subfamily in tissues should be studied. Drug metabolizing activities, such as those of 7benzoxyresorufin O-dealkylase, 7-pentoxyresorufin O-dealkylase, 7-ethoxyresorufin O-deethylase, 7ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase [29, 30], bufuralol l'-monooxygenase [14, 16] and spartein monooxygenase [24], have been found in brain. In this study, low levels of the activities of both debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase were found in cerebral cortex. The presence of P450 db in cerebral cortex is consistent with Western blotting data [31]. The finding of the presence of debrisoquine/spartein monooxygenase activities in brain is important for understanding the metabolism of Parkinsonisminducing substances such as MPTP and TIQ. These neurotoxins or chemicals can be metabolized not only in liver but also in brain. Low levels of bufuralol l'-monooxygenase and debrisoquine 4-monooxygenase activities, with a higher ratio, were detected in kidney cortex. This is consistent with the following observations. Firstly, serum from LKM1 patients, who possess autoantibodies for P450 dbl (IID6) [32-34], cross-reacts
4.806 ± 0.446*[4] (100) 0.075 (1.6) 5.522 (114.9) 0.454 (9.4) 0.785 (16.3) 1.114 (23.2) 0.417 ± 0.389 [2] (8.7) 3.364 ± 0.771 [2] (70.0) 5.116 (106.5) --
--
--
ND
ND
ND
ND** 0.076 (90.5)
0.084 (100)
31.343 -+ 3.000[4] (100) 4.884 (15.6) 29.205 (93.2) 13.019 (41.5) 6.312 (20.1) 2.463 (7.9) 5.737 ± 0.158 [2] (18.3) 33.216 +- 0.905 [2[ (106.0) 35.734 (114.0)
Liver
ND 0.168 (82.8) 0.215 (106.0)
0.203 _+ 0.022[2] (100) 0.035 (17.2) 0.192 (94.6) 0.140 (69.0) 0.075 (36.9) 0.009 (4.4)
Kidney cortex
ND 0.103 (91.2) 0.122 (107.6)
0.113 ± 0.01112] (100) 0.016 (14.2) 0.118 (104.4) 0.072 (63.7) 0.028 (24.8) 0.008 (7.1)
Cortex
Medulla
ND 0.076 (107.0) 0.069 (96.9)
0.071 ± 0.01412] (100) 0.005 (7.0) 0.070 (98.6) 0.022 (31.0) 0.022 (31.0) 0.004 (5.6)
Adrenal
Bufuralol 1'-monooxygenase
--
--
--
0.053 (100) 0.027 (50.9) 0.061 (115.1) 0.036 (67.9) 0.080 (150.9) 0.032 (60.4)
Cerebral cortex
* Enzyme activities are expressed as nmol/hr/mg protein (mean ± SD). The number of samples is indicated in brackets. Within round brackets are the percentage activities relative to control value. ND, not detected; the enzyme activities less than 0.025 and 0.001 nmol/hr/mg protein of microsomes cannot be detected for debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase, respectively. - - Effects of quinidine, mannitol and SOD on enzyme activities of cerebral cortex have not been studied.
Phenylisocyanide (0.1 mM) KCN (1 mM) NaN 3 (5 raM) Metyrapone (1 raM) Sodium cholate (0.5%) Quinidine (0.5 raM) Mannitol (200 mM) SOD (1/tM)
Control
Liver
Cerebral cortex
Debrisoquine 4-monooxygenase
Table 3. Effects of inhibitors, hydroxyl radical scavenger and SOD on the enzyme activities of debrisoquine 4-monooxygenase and bufuraloi l'-monooxygenase in microsomes of various tissues from male cattle
t~
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.=.
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1918
Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
with microsomal protein in kidney cells as well as the high levels in liver ceils [35, 36]. Secondly, in rat kidney, mRNAs for IID2 and IID3 were detected in considerably larger amounts than those of IID1 (which exhibits debrisoquine 4-monooxygenase activity) and IID5 [37]. Therefore, the higher bufuraloi l'-monooxygenase activity ratio in kidney cortex can be explained by a higher ratio of the db2 type protein. In lung, low levels of debrisoquine 4-monooxygenase and bufuralol monooxygenase activities were detected. However, none of the rat P450 IID forms were detected in intestine or lung [11]. This might be either due to the species difference or to the different sensitivities of the methods used. The role of P450dbl in lung might be related to susceptibility to lung cancer. A positive relationship between lung cancer and extensive metabolism of debrisoquine has been reported [13]. The physiological roles of debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities in small intestine and spleen are not clear, however, they are most likely involved in drug metabolism. In the adrenocortex, some drug metabolizing activities, such as those of benzphetamine Ndemethylase [38], ethylmorphine demethylase [39], benzo(a)pyrene hydroxylase [39--42] and aryl hydrocarbon hydroxylase [43], have been detected, including P450s, i.e. IA1, IIA1, IIB1, IIC7 and IIIA1, by Western blotting [44]. The presence of mRNAs for the P450IID subfamily in the adrenocortex was confirmed by means of in situ hybridization and by amplifying c D N A by polymerase chain reaction [45]. No detectable P450 activity has been reported in the adrenomedulla of cattle [46] but we detected a low level of the bufuralol l'-monooxygenase reaction, which was catalysed by P450. This enzymatic activity was not due to contamination by adrenocortex, because the low level of contamination, which if any, is several per cent at the maximum, cannot explain the difference in bufuralol l'-monooxygenase activity between adrenal cortex and medulla. Moreover, the signal obtained on in situ hybridization with a P450IID c D N A probe in the boundary regions of the adrenal cortex and medulla showed approximately the same ratio for bufuralol activity [45]. This is the first evidence for P450 catalysing activity in microsomes from adrenomeduUa. AcknowledgementsbWe are grateful to Dr H. Fukui for
providing the debrisoquine, 4-OH debrisoquine, bufuralol and I'-OH bufuralol, and to Dr H. Kawaguchi of this medical school for supplying the rabbit tissues. We also thank Miss Maki Tsujita and Mr Shuhei Tomita for their help with the preparation of microsomes from tissues. REFERENCES
1. Larrey D, Distlerath LM, Dannan GA, Wilkinson GR and Guengerich FP, Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism. Biochemistry 23: 2787-2795, 1984. 2. Distlerath LM, Reilly PEB, Martin MV, Davis GG,
Wilkinson GR and Guengerich FP, Purification and characterization of the human liver cytochromes P450 involved in debrisoquine 4-hydroxylation and phenacetin o-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J Biol Chem 260: 9057-9067, 1985. 3. Gut J, Catin T, Dayer P, Kronbach T, Zanger U and Meyer UA, Debrisoquine/sparteine-type polymorphism of drug oxidation. J Biol Chem 2,61: 11734-11743, 1986. 4. Fonne-Pfister R and Meyer UA, Xenobiotic and endobiotic inhibitors of cytochrome P-450dbl function, the target of the debrisoquine/sparteine type polymorphism. Biochem Pharmacol 37: 3829--3835, 1988. 5. Ged C, Umbenhauer DR, Bellew TM, Bork RW, Srivastava PK, Shinriki N, Lloyd RS and Guengerich FP, Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase. Biochemistry 27: 6929--6940, 1988. 6. Romkes M, Faletto MB, Blaisdell JA, Raucy JL and Goldstein JA, Cloning and expression of complementary DNAs for multiple members of the human cytochrome P450IIC subfamily. Biochemistry 30: 3247-3255, 1991. 7. Yasumori T, Yamazoe Y and Kato R, Cytochrome P450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogenous P450IIC species expressed in yeast. J Biochem 109: 711717, 1991. 8. Matsunaga E, Zeugin T, Zanger UM, Aoyama T, Meyer UA and Gonzatez FJ, Sequence requirements for cytochrome P-450IID1 catalytic activity. J Biol Chem 2,65: 17197-17201, 1990. 9. Gonzalez FJ, Matsunaga T, Nagata K, Meyer UA, Nebert DW, Pastewka J, Kozak C, Gillette J, Gelboin HV and Hardwick JP, Debrisoquine 4-hydroxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis of the DA rat polymorphism. DNA 6: 149161, 1987. 10. Kronbach T, Mathys D, Gut J, Catin T and Meyer UA, High-performance liquid chromatographic assays for bufuralol l'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan o-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver. Anal Biochem 162: 24--32, 1987. 11. Gonzalez FJ, The molecular biology of cytochrome P450s. Pharmacol Rev 40: 243-288, 1988. 12. Gonzalez FJ, Skoda RC, Kimura S, Umeno M, Zanger UM, Nebert DW, Gelboin HV, Hardwick JP and Meyer UA, Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature 331: 442-446, 1988. 13. Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M and Wolf CR, Identification of the primary gene defect at the cytochrome Pds0 CYP2D locus. Nature 347: 773-776, 1990. 14. Fonne-Pfister R, Bargetzi MJ and Meyer UA, MPTP, the neurotoxin inducing Parkinson's disease, is a potent competitive inhibitor of human and rat cytochrome P450 isozymes (P450bufI, P450dbl) catalyzing debrisoquine 4-hydroxylation. Biochem Biophys Res Commun 148: 1144-1150, 1987. 15. Lee EDJ and Moochhala S, Tissue distribution of bufuralol hydroxylase activity in Sprague--Dawley rats. Life Sci 44: 827-830, 1989. 16. Ohta S, Tachikawa O, Makino Y, Tasaki Y and Hirose M, Metabolism and brain accumulation of tetrahydroisoquinoline (TIQ) a possible Parkinsonism inducing substance, in an animal model of a poor debrisoquine metabolizer. Life Sci 46: 599--605, 1990.
Tissue distribution of debrisoquine/bufuralol monooxygenase activities 17. Shahi GS, Moochhala SM, Das NP, Sunamoto J and Tajima K, The neurotoxin 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP) induces changes in the heme spin state of microsomal cytochrome P-450. Biochem lnt 22: 895-902, 1990. 18. Mitoma C, Posner HS, Reitz HC and Udenfriend S, Enzymatic hydroxylation of aromatic compounds. Arch Biochem Biophys 61: 431-441, 1956. 19. Icbikawa Y and Yamano T, Electron spin resonance of microsomal cytochromes: correlation of the amount of CO-binding species with so-called microsomal Fex in microsomes of normal tissues and liver microsomes of sudan III treated animals. Arch Biochem Biophys 121: 742-749, 1967. 20. Veeger C, DerVartanian V and Zeylemaker WP, Succinate dehydrogenase. Methods Enzymol 13: 8190, 1969. 21. Gornail AG, Bardawill CJ and David MM, Determination of serum proteins by means of the biuret reaction. J Biol Chem 177: 751-766, 1949. 22. Ingeiman-Sundberg M, Kaur H, Terelius Y, Persson JO and Halliwell B, Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Biochem J 276: 753-757, 1991. 23. Boobis AR, Murray S, Hampden CE and Davis DS, Genetic polymorphism in drug oxidation: in vitro studies of human debrisoquine 4-hydroxylase and bufuralol l'-hydroxylase activities. Biochem Pharmacol 34: 65--71, 1985. 24. Tyndale RF, Sunahara R, Inaba T, Kalow W, Gonzalez FJ and Niznik HB, Neuronal cytocbrome P450IID1 (Debrisoquine/Sparteine-type): potent inhibition of activityby (-)-cocaine and nucleotide sequence identity to human hepatic P450 gene CYP2D6. Mol Pharmacol 40: 63-68, 1991. 25. Ichikawa Y and Yamano T, The electron spin resonance and absorption spectra of microsomal cytochrome P450 and its isocyanide complexes. Biochim Biophys Acta 153: 753-765, 1968. 26. Ichikawa Y and Yamano T, Reconversion of detergent and sulfhydryl reagent produced P-420 to P-450 by polyois and glutathione. Biochim Biophys Acta 131: 490-497, 1967. 27. White RE and Coon MJ, Heine ligand replacement reactions of cytochrome P-450. J Biol (?hem 257: 30733083, 1982. 28. Yoshida Y, Imai Y and Hashimoto-Yutsudo C, Spectrophotometric examination of exogenous-ligand complexes of ferric cytochrome P-450. Characterization of the axial ligand trans to thiolate in the native ferric low-spin form. J Biochem 91: 1651-1659, 1982. 29. Perrin R, Minn A, Ghersi-Egea JF, Grassiot MC and Siest G, Distribution of cytochrome P450 activities towards aikoxyresorufin derivatives in rat brain regions, subcellular fractions and isolated cerebral microvessels. Biochem Pharmacol 40: 2145-2151, 1990. 30. Dhawan A, Parmar D, Das M and K Seth P, Cytochrome P-450 dependent monooxygenases in neuronal and glial cells: Inducibility and specificity. Biochem Biophys Res Commun 170: 441--447, 1990. 31. Niznik HB, Tyndale RF, Sallee FR, Gonzalez FJ, Hardwick JP, Inaba T and Kalow W, The dopamine transporter and cytochrome P450IID1 (Debrisoquine 4-hydroxylase) in brain: Resolution and identification
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of two distinct [3H]GBR-12935binding proteins. Arch Biochem Biophys 276: 424--432, 1990. 32. Gueguen M, Meunier-Rotival M, Bernard O and Alvarez F, Anti-liver kidney microsome antibody recognizes a cytochrome P450 from the IID subfamily. J Exp Med 168: 801-806, 1988. 33. Zanger UM, Hauri HP, Loeper J, Homberg JC and Meyer UA, Antibodies against human cytochrome P450dbl in autoimmune hepatitis type II. Proc Natl Acad Sci USA 85: 8256-8260, 1988. 34. Manns MP, Johnson EF, Griffin KJ, Tan EM and Sullivan KF, Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450dbl. J Clin Invest 83: 1066-1072, 1989. 35. Rizzeto M, Francesco BB and Doniach D, Characterization of the microsomal antigen related to a subclass of active chronic hepatitis. Immunology 26: 589-601, 1974. 36. Storch W, Cossel L and Dargel R, The immunoelectronmicroscopical demonstration of antibodies against endoplasmicreticulum (microsomes)in chronic aggressive hepatitis and liver cirrhosis. Immunology 32: 941945, 1977. 37. Matsunaga E, Zanger UM, Hardwick JP, Gelboin HV, Meyer UA and Gonzalez FJ, The Cyp2D gene subfamily: Analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats. Biochemistry 28: 7349-7355, 1989. 38. Hiwatashi A and Ichikawa Y, Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-1inked mixed function oxidase system) of bovine adrenocortical microsomes. Biochim Biophys Acta 664; 33--48, 1981. 39. Greiner JW, Rumbaugh RC, Kramer RE and Colby liD, Relation of canrenone to the actions of spironolactone on adrenal cytochrome P-450-dependent enzymes. Endocrinology 103: 1313-1320, 1978. 40. AIvares AP and Kappas A, Heterogeneity of cytochrome P-450sinduced by polychlorinated biphenyls. J Biol Chem 252: 6373--6378, 1977. 41. Colby HD, Johnson PB, Pope MR and Zulkoski JS, Metabolism of benzo[a]pyrene by guinea pig adrenal and hepatic microsomes. Biochem Pharmaco131: 639-646, 1982. 42. Dao TL and Yogo H, Effects of polynuclear aromatic hydrocarbons on benzpyrene hydroxylase activity in rats. Proc Soc Exp Biol Med 116: 1048-1050, 1964. 43. Guenthner TM, Nebert DW and Menard RH, Microsomai aryl hydrocarbon hydroxylase in rat adrenal: regulation by ACTH but not by polycyclic hydrocarbons. Mol Pharmacol 15: 719-728, 1979. 44. Otto S, Marcus C, Pidgeon C and Jefcoate C, A novel adrenocorticotropin-inducible cytochrome P450 from rat adrenal microsomes catalyzes polycylic aromatic hydrocarbon metabolism. Endocrinology 129: 970-982, 1991. 45. Matsuo Y, Iwahashi K, Kawai Y and Ichikawa Y, Analysis of gene transcripts of P-450 liD subfamily in the bovine adrenal gland. Histochemistry, in press. 46. Ichikawa Y and Yamano T, Cytochrome b-559 in the microsomes of the adrenal medulla. Biochem Biophys Res Commun 20: 263-268, 1965.
0006-2952/92 $5.00 + 0.00 ~ 1992. Pergamon Press Ltd
Printed in Great Britain.
DEBRISOQUINE 4-MONOOXYGENASE A N D BUFURALOL I'-MONOOXYGENASE ACTIVITIES IN BOVINE A N D RABBIT TISSUES YOSHINORI MATSUO,* KAZUHIRO IWAHASHIt and YOSHIYUKIICHIKAWA Department of Biochemistry, Kagawa Medical School, Miki-cho, Kita-gun, Kagawa 761-07, Japan (Received 3 December 1991; accepted 14 February 1992) tissue distributions of debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities in microsomes from bovine and rabbit tissues were analysed. Debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities were found in liver, and at low levels in cerebral cortex, kidney cortex, lung, small intestine and spleen. Other tissues, such as kidney medulla, adrenocortex, adrenomedulla, blood vessels, thyroid gland, heart, ovary, uterus and testis, showed low levels of bufuralol l'-monooxygenase activity but no detectable debrisoquine 4-monooxygenase activity. The bufuralol/debrisoquine monooxygenase activity ratios were higher in kidney and lung, and lower in cerebral cortex and spleen than in liver. Both monooxygenase activities in several bovine tissues including liver were inhibited strongly by phenylisocyanide (0.1mM) and quinidine (0.5mM), moderately by metyrapone (1 raM), and not at all by KCN (1 mM). NuNs (5 raM) and sodium cholate (0.5% w/v) inhibited debrisoquine 4-monooxygenase activity strongly and moderately, but bufuralol l'-monooxygenase activity moderately and strongly, respectively. No effect of a hydroxyl radical scavenger or of superoxide dismutase on either monooxygenase activity was observed. It was concluded from these results, as well as the NADPH dependency of the reactions, that the two monooxygenase reactions observed in these tissues were catalysed by cytochrome P450s.
Abstract--The
Over 25 drugs and chemicals, including debrisoquine, bufuralol, sparteine and dextromethorphan, are metabolized by cytochrome P450 IID [1-4]. Some members of the cytochrome P450 (P450,) subfamily differing only slightly in amino acid sequence show different substrate specificities [5-8], and are expressed differently in various tissues and at various ages [9, 10]. In rat liver, two members of the P450 IID subfamily, the dbl and db2 proteins, were identified [9]. The former exhibits both debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities (dbl type), and the latter bufuralol l'monooxygenase activity but not debrisoquine 4monooxygenase activity (db2 type) [9]. Similarly, in human liver, the dbl type (P450 buff) and db2 type (P450 bufII) were identified [3, 10]. Therefore, the debrisoquine 4-monooxygenase reaction is catalysed by P450 dbl, while the bufuralol l'-monooxygenase reaction is catalysed by more than two forms of P450. Usually, several P450s are responsible for the metabolism of a certain drug or chemical [11]. However, the substrate specificity of debrisoquine * Corresponding author: Y. Matsuo. Tel. (81) 878-985111; FAX (81) 878-98-7109. t Present address: Department of Neuropsychiatry, Kagawa Medical School, Kagawa, Japan. Abbreviations: P450, cytochrome P450; LKM, liver kidney microsomes; MPTP, 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine; TIQ, tetrahydroisoquinoline; G6P, D-glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase. § Tsuneoka Y, Matsuo Y and Ichikawa Y. Characterization and microheterogeneity of cytochrome P-450 IID subfamily from bovine liver: nucleotide sequence and classification of the members of cytochrome P-450 IID subfamily. Manuscript in preparation.
is marked as it is apparently only metabolized by P450dbl and individuals with defects in the gene for P450db1 were poor metabolizers of debrisoquine [12, 13]. Although bufuralol l'-monooxygenase activity has been measured in several rat tissues, such as liver, kidney, brain, lung and spleen [14,15], the tissue distribution of debrisoquine 4-monooxygenase activity, which is important for the metabolism of Parkinsonism-inducing substances such as 1-methyl4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) and tetrahydroisoquinoline (TIQ) [14, 16, 17], has not been reported and could be different from the distribution of bufuralol l'-monooxygenase activity. In this study bovine and rabbit tissues were used because of their size, especially small organs such as adrenal glands and various parts of the brain which renders the preparation of microsomes, the purification and extraction of RNA, and the detection of localized proteins or mRNAs easier. We have cloned and analysed cDNA clones of the P450 IID subfamily from bovine liver, and reported the presence of microheterogeneity§. To study the functions and expressional control of members of the P450 IID subfamily, the tissue distributions of debrisoquine 4-monooxygenase and bufuralol l'monooxygenase activities in bovine and rabbit tissues were examined in this study. MATERIALS AND METHODS
Chemicals. Debrisoquine sulfate, 4-hydroxydebrisoquine sulfate, bufuralol hydrochloride (race- [(tert- butylamino)methyl]- 7-ethyl-2-benzofuranmethanol hydrochloride) and l'-hydroxybufuralol were kindly supplied by Dr H. Fukui (Department
1911
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Y. MATSUO, K. IWAHASHI and Y. ICHIKAWA
of Pharmacology, Medical School, Osaka University). D-Glucose 6-phosphate (G6P) and glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides were purchased from Oriental Yeast Co. Ltd, Japan. N A D P H and phenylmethyl sulfonyl fluoride were purchased from the Sigma Chemical Co. (St Louis, MO, U.S.A.). Superoxide dismutase (SOD) was purchased from Toyobo Co. Ltd, Japan. Materials. The cattle (Holstein-Friesian, 1.5 years old) were obtained from a local slaughterhouse. The rabbits (Japan, white, 6 months old) had been given a normal diet and water ad lib. in the laboratory. Fresh tissues from the animals were kept on ice and transported to the laboratory within 1 hr. They were perfused with ice-cold 0.15 M KCI to remove blood. They were then rapidly and carefully cleared of connective tissue, fat and major blood vessels. Preparation of microsomes. Microsomal fractions from bovine and rabbit tissues were prepared by the method of Mitoma et al. [18]. Care was taken to remove hemoglobin as much as possible, as described previously [19]. Contamination of the microsomes of various tissues by mitochondria was found to be less than 5% on a protein basis, as judged from the succinate dehydrogenase activity [20]. Using bovine serum albumin as a standard, the protein concentrations in microsomal fractions were measured by the biuret reaction method [21] in the presence of 0.25% (w/v) deoxycholic acid, which was added to remove turbidity from the sample. The increased absorption at 540 nm in the biuret reaction due to heine in the test samples was avoided by using a solution containing 3.4% (w/v) NaOH, KI (1 g/500 mL) and potassium sodium tartrate 3 g/ 500 mL). The optical absorption at 25° was measured, using cuvettes of 1 cm optical path, with a Cary recording spectrophotometer, model 17D.
Assaying of debrisoquine 4-monooxygenase and bufuralol l '-monooxygenase activities. The enzymatic reactions and chromatographic conditions reported by Kronbach et al. [10] were slightly modified and used for the activity assays. In the NADPHregenerating system, G6P and G6PDH were used instead of sodium isocitrate and isocitrate dehydrogenase. The reaction mixture, which contained microsomal protein (0.3-2 mg), a N A D P H regenerating system without N A D P H (5 mM G6P, 2.5U G6PDH and 4 m M MgC12) and 0.5mM substrate (debrisoquine or bufuralol concentrations of more than three times the Km values reported for human and rat P450db) in 0.05 M sodium phosphate buffer (pH7.4), was preincubated for 3min at 37°. In the reactions with pancreas microsomes, phenylmethyl sulfonyl fluoride was added to 0.5 mM to inhibit protease activity. The reaction was started by the addition of N A D P H (final concentration, 1.6 mM), followed by aerobic incubation for 60 min at 37°. Under these conditions, the enzymatic reactions were linear with time for at least 80 min and microsomal protein concentrations up to 2 mg/ mL. Only when the presence of a trace of activity was checked, the mixture was incubated for 2 hr at 37° . The final volume of the reaction mixture was 1 mL. The reaction was stopped by the addition of 100/~L of 60% HC104 (w/v). Denatured protein
was removed by centrifugation. The enzymatic production of 4-hydroxydebrisoquine or l'-hydroxybufuralol was measured by HPLC (Waters 510), with fluorescence detection, as described by Kronbach et al. [10]. The column used was of Cosmosil 5C-18 (Nacalai Tesque, 4.6 x 250 mm) for both activity assays. In most cases, the retention times of l'-hydroxybufuralol and 4-hydroxydebrisoquine were 4.2 and 9.5 min, respectively. In some cases, however, overlapping peaks of products had to be separated by adjusting the retention time of 4hydroxydebrisoquine in the range of 9.3 to 18.8 rain. In the inhibition experiment, phenylisocyanide, KCN, NaN3, metyrapone, sodium cholate and quinidine were added to reaction mixtures to final concentrations of 0.1 mM, 1 raM, 5 mM, 1 mM, 0.5% (w/v) and 0.5 raM, respectively. Mannitol, a hydroxyl radical scavenger [22], and SOD were added to a final concentration of 200 mM and 1/~M, respectively. RESULTS
Debrisoquine 4-monooxygenase activity in various tissues Debrisoquine 4-monooxygenase and bufuralol l'monooxygenase activities in the microsomes of various tissues from male and female cattle, and male and female rabbits were analysed. The results are summarized in Table 1. A high level of debrisoquine 4-monooxygenase activity was detected in liver from both cattle and rabbits, and a low level in the cerebral cortex of cattle, lung of cattle and rabbits, small intestine of rabbits, spleen of cattle and kidney cortex of rabbits. No significant sex differences were detected for the debrisoquine 4-monooxygenase activity in the rabbit tissues studied. Debrisoquine 4-monooxygenase activity in male rabbit liver was eight times higher than in male bovine liver (35.5-+ 6.5 vs 4.4 +-0.6nmol/hr/mg protein) The order of activity in rabbit liver was the same as in rat liver [9], and that in bovine liver was the same as in human liver [10, 23]. Debrisoquine 4-monooxygenase activity in bovine cerebral cortex was about 3% of the liver value. Because of the low level, the activity could not be detected when rabbit microsomes from whole cerebrum were analysed as to enzyme activity. None of the other brain parts, i.e. midbrain, cerebellum and medulla oblongata, showed detectable activity. This suggested that debrisoquine 4-monooxygenase activity is higher in the cerebral cortex region of the cerebrum than any other brain parts studied. This is consistent with the regional distribution of spartein monooxygenase activity in the brain [24]. Debrisoquine 4-monooxygenase activity in bovine kidney cortex could not be detected, but low levels of activity, 1.5% and 0.8% of the liver value, were found in male and female rabbit kidney cortex, respectively. In lung, activity levels of 0.7% and 0.5% of the liver value were observed in male cattle and female rabbits, respectively. The activities in small intestine, 0.3% of the liver value, and spleen, 0.6% of the
Tissue distribution of debrisoquine/bufuralol monooxygenase activities
1913
Table 1. Debrisoqnine 4-monooxygenase and bufuralol l'-monooxygenase activities in microsomes of various tissues from bovine and rabbit Debrisoquine 4-monooxygenase (nmol/hr/mg protein) (%)
Bufuralol l'-monooxygenase (nmol/hr/mg protein) (%)
Male Male Female
4.417 -+ 0.551(2) 35.526 - 6.491(2) 23.812
100 100 100
34.397 ± 4.318(2) 66.596 65.729
100 100 100
Bovine Rabbit Rabbit Rabbit Rabbit
Male Male Female Male Female
ND(3) 0.526 0.187 ND(2) ND(2)
1.48 0.79
0.392 -4-0.328(3) 1.653 -*- 1.955(5) 2.933 - 2.007(3) 0.540 - 0.592(2) 0.144 _-+0.172(2)
1.14 2.48 4.46 0.82 0.22
Bovine Bovine
Male Male
ND(3) ND(2)
0.133 ± 0.035(3) 0.075 ± 0.006(2)
0.39 0.22
Bovine Rabbit Rabbit Rabbit Rabbit Rabbit
Male Male Female Male Male Female
0.132 -+ 0.083(3) ND ND ND ND ND
0.085 -+ 0.050(3) 0.018 0.026 0.017 0.010 0.009
0.25 0.03 0.04 0.03 0.02 0.01
Rabbit Bovine Rabbit Bovine Rabbit Bovine Rabbit Rabbit Rabbit Bovine Rabbit Bovine Rabbit Bovine Rabbit Rabbit Rabbit
Male Male Female Male Female Male Male Female Female Male Female Male Female Female Female Female Male
ND 0.032 0.127 ND 0.070 ND ND ND ND ND ND 0.028 ND ND ND ND ND
0.010 0.196 0.843 0.024 0.076 0.005 0.053 0.063 0.038 0.022 0.039 0.067 0.016 0.019 0.017 0.034 0.165
0.02 0.57 1.28 0.07 0.12 0.01 0.08 0.10 0.06 0.06 0.06 0.19 0.02
Tissues
Animals
Liver
Bovine Rabbit Rabbit
Kidney Cortex Medulla Adrenal gland Cortex Medulla Brain Cerebral cortex Cerebrum Midbrain Cerebellum Medulla oblongata Lung Small intestine Blood vessels Thyroid gland Heart Pancreas Spleen Ovary Uterus Testis
Sex
2.99
0.72 0.53 0.29
0.63
0.03 0.05 0.25
Values are means -+ SD. Within parentheses are the number of samples from different animals. ND, not detected; the enzyme activities less than 0.025 and 0.001 nmol/hr/mg protein of microsomes cannot be detected for debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase, respectively.
liver value, were detected in only one of the two species studied, respectively. There was no detectable debrisoquine 4-monooxygenase activity in kidney medulla, adrenocortex, adrenomedulla, blood vessels, thyroid gland, heart, pancreas, ovary, uterus or testis. To check for the presence of a trace of debrisoquine 4-monooxygenase activity in bovine kidney cortex and adrenocortex, the reaction mixtures were incubated longer (2 hr). However, 4-hydroxydebrisoquine, the product of the reaction, could not be detected in these tissues. Low levels of other unknown metabolites were detected in microsomes of the adrenocortex (Fig.
1).
Bufuralol l'.monooxygenase activity in various tissues Bufuralol l'-monooxygenase activities in various tissues are shown in Table 1 and representative
chromatographic patterns in Fig. 2. There was a general tendency that the higher was the debrisoquine 4-monooxygenase activity in certain tissues, the higher was the bufuralol l'-monooxygenase activity in the same tissue. High levels of bufuralol l ' monooxygenase activity were found in liver, and low levels in kidney cortex, adrenocortex, adrenomedulla, cerebral cortex, lung, small intestine and spleen. Traces of activity were found in cerebrum, midbrain, cerebellum and medulla oblongata from rabbit brain, and blood vessels, thyroid gland, heart, pancreas, ovary, uterus and testis. The activity levels in liver from male cattle, and male and female rabbits were 34.4, 66.6 and 65.7 nmol/hr/mg protein, respectively. The bufuralol l'-monooxygenase activity levels in kidney cortex from male cattle, and male and female rabbits were 1.1%, 2.5% and 4.5% of the liver values, respectively.
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Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
4-OH
(A) Bovine Kidney
(C) Bovine Adrenocortex
(a) Rabbit Liver
(D) 4-OH Debrisoquine
F Fig. 1. Chromatograms of debrisoquine metabolites with a longer incubation period (2 hr). (A) Incubation of kidney cortex microsomes from male cattle with debrisoquine. (B) Liver microsomes from male rabbits. 4-OH, 4-hydroxydebrisoquine. (C) Adrenocortex microsomes from male cattle. (D) Standard 10.4 nmol of 4-hydroxydebrisoquine. Six hundred microlitres of the reaction mixture were injected in each case.
Bufuralol l'-monooxygenase activity was found in adrenal cortex and medulla, 0.4% and 0.2% of the liver value, respectively. In adrenal cortex and medulla, no debrisoquine 4-monooxygenase activity was detected, suggesting the presence of db2 type I)450 in adrenal cortex and medulla.
A low level of bufuralol l'-monooxygenase activity was found in cerebral cortex (0.3% of the liver value) and very low levels in other brain parts. Like debrisoquine 4-monooxygenase activity in cerebral cortex, bufuralol l'-monooxygenase activity was also higher in the cerebral cortex region of the brain.
Tissue distribution of debrisoquine/bufuralol monooxygenase activities
1915
1 ' -OH
I'-OH
1 ' -01/
I'-OH
1 '-OH
1 ' -OH
1 ' -OR
(A) I'-OH Bufuralol
(R)
(C)
(D)
Rabbit Liver 1 /zl
Bovine Liver 1 ;tl
Bovine Kidney 100 /.zl
(El Bovine Adrenocortex i00 ~ I
(F)
(G)
Bovlne
Bovine
Adrenoaedulla 200 ~ I
Cerebral 200 /.zl
cortex
Fig. 2. Chromatograms of bufuralol metabolites. (A) Forty picomoles of l'-hydroxybufuralol as a standard. (B) Metabolites formed by liver microsomes from male rabbits. One microlitre of the reaction mixture was injected. 1'-OH, l'-hydroxybufuralol. (C) Liver microsomes from male cattle, 1 #L. (D) Kidney cortex microsomes from male cattle, 100/zL. (E) Adrenocortex microsomes from male cattle, 100 #L. (F) Adrenomedulla microsomes from male cattle, 200/zL. (G) Cerebral cortex microsomes from male cattle, 200 #L.
Other tissues which showed low levels of bufuralol l'-monooxygenase activity were lung, small intestine and spleen. These tissues also showed low levels of debrisoquine 4-monooxygenase activity. No significant sex difference was detected for bufuralol l'-monooxygenase activity in rabbits.
Bufuralol/debrisoquine monooxygenase ratio in various tissues The bufuralol l'-monooxygenase/debrisoquine 4monooxygenase activity ratios are shown in Table 2. There was variation in the ratio in various tissues. -In liver, the ratios were 7.8 in male cattle, 1.9 in male rabbit and 2.8 in female rabbit, however, the ratios in kidney cortex were 3.1 in male rabbit and 15.7 in female rabbit. These values were higher than in liver. In lung the ratio in female rabbit, 6.6, was higher than in liver, but the ratio in male cattle, 6.1, was about the same as in liver. In contrast, bovine cerebral cortex and spleen showed lower ratios, 0.6 and 2.4, respectively, than liver.
Effects of inhibitors on debrisoquine 4-monooxygenase and bufuralol l '-monooxygenase activities To confirm the P450 dependent reaction, the dependence of the reaction on N A D P H as an electron donor, the effects of six chemicals, phenylisocyanide, KCN, NAN3, metyrapone, sodium cholate and quinidine, on the debrisoquine 4monooxygenase and bufuralol l'-monooxygenase activities were investigated with microsomes from several bovine tissues, i.e. liver, kidney cortex, adrenocortex, adrenomedulla and cerebral cortex. Phenylisocyanide is known to be a specific inhibitor of P450 [25] and sodium cholate is a denaturing reagent, converting P450 to P420 [26]. The results are shown in Table 3. Debrisoquine 4-monooxygenase activity in liver was strongly inhibited by 0.1 mM phenylisocyanide, 5 mM NaN 3 and 0.5 mM quinidine to 1.6%, 9.4% and 8.7% of the control value, respectively, and moderately by 1 mM metyrapone and 0.5% (w/v) sodium cholate, to 16.3% and 23.2%, respectively,
Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
1916
Table 2. Ratios of bufuralol l'-monooxygenase (But') to debrisoquine 4-monooxygenase(Deb) activities in various tissues Tissues Liver
Animals
Bovine Rabbit Rabbit Kidney cortex Rabbit Rabbit Brain cerebral cortex Bovine Lung Bovine Rabbit Small intestine Rabbit Spleen Bovine
Sex Male Male Female Male Female Male Male Female Female Male
Ratios (Buf/Deb) 7.79 1.87 2.76 3.14 15.68 0.64 6.13 6.64 1.09 2.39
Effects of a hydroxyl radical scavenger and SOD on the enzyme activities To investigate the effects of hydroxyl radicals and OF, a hydroxyl radical scavenger, mannitol, and SOD were added to final concentrations of 200 mM and 1/~M, respectively, and then the enzyme activities in several bovine tissues were measured (Table 3). Although debrisoquine 4-monooxygenase activity in liver in the presence of mannitol was decreased to 70%, bufuralol l'-monooxygenase activity did not change, 82-107% of the control value. SOD did not affect the enzyme activities, 97114% of the control values, in the tissues studied. These results show that neither hydroxyl radicals nor O~ had significant effects on these activity assays. DISCUSSION
but not inhibited by 1 mM KCN. The reaction without NADPH or a substrate showed no detectable debrisoquine 4-monooxygenase activity. A similar activity inhibition pattern was seen in cerebral cortex. Because of the low activity in cerebral cortex, which was just above background, the activity inhibited by phenylisocyanide, NAN3, metyrapone and sodium cholate could not be detected. The activity was not inhibited by KCN, 90.5%, and not detected without NADPH or a substrate. Bufuralol l'-monooxygenase activities in microsomes from the various tissues were inhibited very similarly by these inhibitors, except for cerebral cortex. The activities of various tissues were inhibited strongly by 0.1 mM phenylisocyanide, 0.5% (w/v) sodium cholate and 0.5 mM quinidine, 4-18% of the control value, and moderately by 1 mM metyrapone and 5 mM NaN3, 20--69%. One millimolar KCN did not inhibit the activity, 93-104%. No detectable activity was found without a substrate. However, traces of activity were detected without NADPH, 0.003-0.055 nmol/hr/mg protein, in which case the reaction might be driven by either endogenous NADPH or an enzyme that is independent of NADPH. The activity inhibition pattern in cerebral cortex seemed to be different. However, the activity was inhibited 49.1%, 32.1% and 39.6% by 0.1 mM phenylisocyanide, 5mM NaN2 and 0.5% sodium cholate, respectively, but not at all by 1 mM KCN or 1 mM metyrapone. These results of inhibition experiments, i.e. that the activities in several tissues were strongly inhibited by phenylisoeyanide, inhibited very similarly by several inhibitors and depended on NADPH, confirmed that the low level of the debrisoquine 4monooxygenase reaction in brain, and the bufuralol l'-monooxygenase reactions detected in kidney cortex, adrenal gland and brain, as well as the high level in liver, were catalysed by P450. P450s have been classified into two groups according to their sensitivity to KCN [27, 28]. From the results of the inhibition experiments, it is clear that P450 db belongs to the group insensitive to KCN.
Bufuralol l'-monooxygenase activity has been reported in several rat tissues, such as liver, kidney, brain, adrenal, lung and spleen [15]. The activities were very close to those reported here for cattle and rabbits. Debrisoquine 4-monooxygenase activity in various tissues was also determined and variations in the bufuralol/debrisoquine monooxygenase activity ratio were found. Moreover, the ratios in a single tissue in two species were also different, for example, the level of debrisoquine 4-monooxygenase activity in rabbit liver was eight times higher than that in bovine liver. However, bufuralol l'-monooxygenase activity was not very different in the livers of the two species. The variation in the activity ratio in tissues or species could be explained by either or both of the following. (1) The efficiency of debrisoquine or bufuralol metabolism by a single P450 molecule. (2) The ratio of the expressed proteins, dbl type and db2 type. To clarify this, enzymatic and kinetic parameters, such as Km and Vmax, and the turnover numbers for each type of db protein and the degree of expression of each member of the subfamily in tissues should be studied. Drug metabolizing activities, such as those of 7benzoxyresorufin O-dealkylase, 7-pentoxyresorufin O-dealkylase, 7-ethoxyresorufin O-deethylase, 7ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase [29, 30], bufuralol l'-monooxygenase [14, 16] and spartein monooxygenase [24], have been found in brain. In this study, low levels of the activities of both debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase were found in cerebral cortex. The presence of P450 db in cerebral cortex is consistent with Western blotting data [31]. The finding of the presence of debrisoquine/spartein monooxygenase activities in brain is important for understanding the metabolism of Parkinsonisminducing substances such as MPTP and TIQ. These neurotoxins or chemicals can be metabolized not only in liver but also in brain. Low levels of bufuralol l'-monooxygenase and debrisoquine 4-monooxygenase activities, with a higher ratio, were detected in kidney cortex. This is consistent with the following observations. Firstly, serum from LKM1 patients, who possess autoantibodies for P450 dbl (IID6) [32-34], cross-reacts
4.806 ± 0.446*[4] (100) 0.075 (1.6) 5.522 (114.9) 0.454 (9.4) 0.785 (16.3) 1.114 (23.2) 0.417 ± 0.389 [2] (8.7) 3.364 ± 0.771 [2] (70.0) 5.116 (106.5) --
--
--
ND
ND
ND
ND** 0.076 (90.5)
0.084 (100)
31.343 -+ 3.000[4] (100) 4.884 (15.6) 29.205 (93.2) 13.019 (41.5) 6.312 (20.1) 2.463 (7.9) 5.737 ± 0.158 [2] (18.3) 33.216 +- 0.905 [2[ (106.0) 35.734 (114.0)
Liver
ND 0.168 (82.8) 0.215 (106.0)
0.203 _+ 0.022[2] (100) 0.035 (17.2) 0.192 (94.6) 0.140 (69.0) 0.075 (36.9) 0.009 (4.4)
Kidney cortex
ND 0.103 (91.2) 0.122 (107.6)
0.113 ± 0.01112] (100) 0.016 (14.2) 0.118 (104.4) 0.072 (63.7) 0.028 (24.8) 0.008 (7.1)
Cortex
Medulla
ND 0.076 (107.0) 0.069 (96.9)
0.071 ± 0.01412] (100) 0.005 (7.0) 0.070 (98.6) 0.022 (31.0) 0.022 (31.0) 0.004 (5.6)
Adrenal
Bufuralol 1'-monooxygenase
--
--
--
0.053 (100) 0.027 (50.9) 0.061 (115.1) 0.036 (67.9) 0.080 (150.9) 0.032 (60.4)
Cerebral cortex
* Enzyme activities are expressed as nmol/hr/mg protein (mean ± SD). The number of samples is indicated in brackets. Within round brackets are the percentage activities relative to control value. ND, not detected; the enzyme activities less than 0.025 and 0.001 nmol/hr/mg protein of microsomes cannot be detected for debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase, respectively. - - Effects of quinidine, mannitol and SOD on enzyme activities of cerebral cortex have not been studied.
Phenylisocyanide (0.1 mM) KCN (1 mM) NaN 3 (5 raM) Metyrapone (1 raM) Sodium cholate (0.5%) Quinidine (0.5 raM) Mannitol (200 mM) SOD (1/tM)
Control
Liver
Cerebral cortex
Debrisoquine 4-monooxygenase
Table 3. Effects of inhibitors, hydroxyl radical scavenger and SOD on the enzyme activities of debrisoquine 4-monooxygenase and bufuraloi l'-monooxygenase in microsomes of various tissues from male cattle
t~
O O
O
o
t~
.=.
r~
{3"
o
O
tnr
e~
S.
1918
Y. MATSUO,K. IWAHASHIand Y. ICHIKAWA
with microsomal protein in kidney cells as well as the high levels in liver ceils [35, 36]. Secondly, in rat kidney, mRNAs for IID2 and IID3 were detected in considerably larger amounts than those of IID1 (which exhibits debrisoquine 4-monooxygenase activity) and IID5 [37]. Therefore, the higher bufuraloi l'-monooxygenase activity ratio in kidney cortex can be explained by a higher ratio of the db2 type protein. In lung, low levels of debrisoquine 4-monooxygenase and bufuralol monooxygenase activities were detected. However, none of the rat P450 IID forms were detected in intestine or lung [11]. This might be either due to the species difference or to the different sensitivities of the methods used. The role of P450dbl in lung might be related to susceptibility to lung cancer. A positive relationship between lung cancer and extensive metabolism of debrisoquine has been reported [13]. The physiological roles of debrisoquine 4-monooxygenase and bufuralol l'-monooxygenase activities in small intestine and spleen are not clear, however, they are most likely involved in drug metabolism. In the adrenocortex, some drug metabolizing activities, such as those of benzphetamine Ndemethylase [38], ethylmorphine demethylase [39], benzo(a)pyrene hydroxylase [39--42] and aryl hydrocarbon hydroxylase [43], have been detected, including P450s, i.e. IA1, IIA1, IIB1, IIC7 and IIIA1, by Western blotting [44]. The presence of mRNAs for the P450IID subfamily in the adrenocortex was confirmed by means of in situ hybridization and by amplifying c D N A by polymerase chain reaction [45]. No detectable P450 activity has been reported in the adrenomedulla of cattle [46] but we detected a low level of the bufuralol l'-monooxygenase reaction, which was catalysed by P450. This enzymatic activity was not due to contamination by adrenocortex, because the low level of contamination, which if any, is several per cent at the maximum, cannot explain the difference in bufuralol l'-monooxygenase activity between adrenal cortex and medulla. Moreover, the signal obtained on in situ hybridization with a P450IID c D N A probe in the boundary regions of the adrenal cortex and medulla showed approximately the same ratio for bufuralol activity [45]. This is the first evidence for P450 catalysing activity in microsomes from adrenomeduUa. AcknowledgementsbWe are grateful to Dr H. Fukui for
providing the debrisoquine, 4-OH debrisoquine, bufuralol and I'-OH bufuralol, and to Dr H. Kawaguchi of this medical school for supplying the rabbit tissues. We also thank Miss Maki Tsujita and Mr Shuhei Tomita for their help with the preparation of microsomes from tissues. REFERENCES
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